Several cell surface associated proteins of the Staphylococci and Streptococci involved in microbial adhesion to different host tissues and considered to be important factors in bacterial pathogenesis have been identified in the last decade (see Patti, J. M., Allen, B. L., McGavin, M. J. and Hook, M., MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues [1994] Annu.Rev.Microbiol. 48, 585-617.).
The LPXTG motif has been identified as characteristic of surface proteins in Grain-positive bacteria (Navarre, W. W. and Schneewind,O. [1994] Molecular Microbiology 14(1) 115-121); Fischetti et al. [1990] Mol. Microbiol. 4 1603-5; Schneewind et al. [1993] EMBO J. 4803-4811).
Navarre, W. W. and Schneewind, O. [1994] demonstrate that the position of the LPXTG motif in a cell surface protein dictates that region of the protein which is anchored to the cell wall and the proportion of the N-terminal fragment which no longer resides in the cytoplasm.
Different approaches have been put forward to address such proteins from Staphylococcus aureus as antibacterial targets, e.g. fibronectin binding proteins (EP0294349, EP0397633, WO94/18327), fibrinogen binding protein (WO94/06830), collagen binding protein (WO92/07002) and bone sialoprotein binding protein (WO94/13310). The binding proteins or binding fragments thereof are used as antibacterial agents to block binding of the organism to host tissue, as vaccines to raise antibodies to the organism in the host animal or as antigens to raise therapeutic antibodies which can be used to block binding of the organism to host tissue.
Recently several novel approaches have been described which purport to follow global gene expression during infection (Chuang, S. et al. [1993] Global Regulation of Gene Expression in Escherichia coli J. Bacteriol. 175, 2026-2036, Mahan, M.J. et al. [1993] Selection of Bacterial Virulence Genes That Are Specifically Induced in Host Tissues SCIENCE 259, 686-688, Hensel, M. et al. [1995] Simultaneous Identification of Bacterial Virulence Genes by Negative Selection SCIENCE 269, 400-403). These new techniques have so far been demonstrated with gram negative pathogen infections and not with infections with gram positives presumably due to the much slower development of global transposon mutagenesis and suitable vectors needed for these strategies in these organisms, and in the case of that process described by Chuang, S. et al.[1993] the difficulty of isolating suitable quantities of bacterial RNA free of mammalian RNA derived from the infected tissue to furnish bacterial RNA labelled to sufficiently high specific activity. The present invention employs a novel technology to determine gene expression in the pathogen at different stages of infection of the mammalian host. A novel aspect of this invention is the use of a suitably labelled oligonucleotide probe which anneals specifically to the bacterial ribosomal RNA in Northern blots of bacterial RNA preparations from infected tissue. Using the more abundant ribosomal RNA as a hybridisation target greatly facilitates the optimisation of a protocol to purify bacterial RNA of a suitable size and quantity for RT-PCR from infected tissue.
A suitable oligonucleotide useful for applying this method to genes expressed in Staphylococcus aureus is
5'-gctcctaaaaggttactccaccggc-3'
Use of the technology of the present invention enables identification of bacterial genes transcribed during infection, inhibitors of which would have utility in anti-bacterial therapy. Specific inhibitors of such gene transcription or of the subsequent translation of the resultant mRNA or of the function of the corresponding expressed proteins would have utility in anti-bacterial therapy.